Method for preparing malonate methylidene nanoparticles, nanoparticles optionally containing one or several biologically active molecules

ABSTRACT

The invention relates to a method for the preparation of nanoparticles formed from a random polymer of at least one compound of formula (I)  
                 
 
     group  
     R 1  and R 2 , identical or different, represents a linear or branched C 1 -C 6  alkyl group;  
     n= 1, 2, 3, 4  or  5,    
     characterised in that the monomer is dissolved beforehand in a water-miscible aprotic organic solvent forming, with the polymerization medium, a non-solvent mixture of the polymer formed.  
     The invention also relates to said nanoparticles, which optionally contain one or more biologically active molecules, and the pharmaceutical compositions containing them.

[0001] The present invention relates to a novel method for thepreparation of nanoparticles formed from a polymerised methylidenemalonate compound, said nanoparticles, optionally containing one or morebiologically active molecules, as well as to pharmaceutical compositionscontaining them.

[0002] “Nanoparticles” is understood as meaning sub-micron particleshaving a diameter of less than about 500 nanometres. Nanoparticlesformed by emulsion polymerisation of an alkyl cyanoacrylate aredescribed in the EP 0 007 895 patent. The method used in the preparationof these alkyl cyanoacrylate particles relies on the (anionic)polymerisation of the monomer which takes place spontaneously and in anaqueous medium. The preparation which follows the same principle(anionic emulsion polymerisation) of nanoparticles constituted of amethylidene malonate polymer is described notably in F. Lescure et al,Pharm. Res., 1994, 11, 1270-1276. These monomers, whose preparation isdescribed in the EP 0 283 364 patent, have a structure close to that ofthe cyanoacrylates but the nitrile function of the latter is replacedwith an ester or an ester ester. Like the cyanoacrylates, theypolymerise in the cold in an aqueous medium and can be biodegradable.

[0003] However, the methylidene malonate nanoparticles thus obtainedpossess certain drawbacks.

[0004] In fact, the emulsion polymerisation of methylidene malonates inthe form of nanoparticles leads, in aqueous phase and at slightly acidpH, to the formation of oligomers, mainly of the trimer or tetramertype, which are highly biodegradable. These molecular species arepartially hydrosoluble, such that the dispersion of these nanoparticlesin an aqueous medium leads to their solubilisation and to the rapid lossof the particle structure (P. Breton et al., Eur. J. Pharm. Biopharm.,1996, 42, 95-103). When a biologically active molecule is associatedwith the methylidene malonate nanoparticles, is therefore possible forthe molecule to be released very rapidly after the administration,following the effect of dilution in the circulatory current which bringsabout the rapid solubilisation of the oligomers which form the particlematrix, before eventually arriving at the site of action of the activeprinciple.

[0005] Certain experiments have shown that the polymerization at basicpH enabled the formation of polymers of higher molecular masses whilemaintaining the size of the nanoparticles. However, such syntheses arecharacterised by:

[0006] the impossibility of obtaining polymers of Mw<10 000, and afortiori Mw<8000. constituting individualised nanoparticles, withoutforming aggregates and without the significant presence of oligomericspecies.

[0007] the impossibility of constituting polymers of Mw>20 000 and afortiori of higher Mw, at high pH (pH>7) without the inevitableformation of aggregates which render the intravascular administration ofthese preparations impossible.

[0008] “Mw” is understood as meaning the mass average molecular mass (oraverage molecular mass) defined as: Mw=Σni.Mi² /Σni.Mi and Mp means themolecular mass of the quantitatively major species.

[0009] In the rest of the description, the molecular mass is expressedin polystyrene equivalents (Ep).

[0010] This preparative method is therefore not suitable if it isdesired to prepare methylidene malonate nanoparticles constituted of:

[0011] polymers of average molecular mass between about 5000 and 10000,notably about 8000,

[0012] polymers of average molecular mass greater than 20000, withoutforming aggregates.

[0013] The present invention therefore consists of the preparation ofmethylidene malonate nanoparticles having a diameter of less than 500nm, in particular 100 to 500 nm. formed from homogeneous molecularspecies of wide-ranging masses (Mw between about 2000 and 80000). Theprinciple consists in dissolving the monomer in a water-miscible aproticorganic phase but which, under the conditions of preparation of thenanoparticles, forms, with the aqueous polymerisation medium, anon-solvent mixture of the polymer formed.

[0014] “Aprotic organic phase” or “aprotic organic solvent”, isunderstood as meaning an organic phase or a solvent without labileproton which is capable of initiating an anion.

[0015] The advantages of this preparative method according to theinvention are numerous:

[0016] it enables a more homogeneous dispersion of the monomer in thepolymerisation medium,

[0017] it makes use of non-chlorinated solvents which are easy toevaporate since they are volatile,

[0018] it prevents the formation of polymer aggregates,

[0019] it gives rise to high polymerisation yields,

[0020] it enables the constitution of polymers of homogeneouswide-ranging molecular mass (Mw about 2000 to 100000, notably about 2000to 80000) in forming nanoparticles having a diameter of less than 500nm.

[0021] Furthermore, the method enables the use of dispersing agents suchas non-ionic surfactants or colloid protecting polymers, which leads toparticles having flexible surface properties.

[0022] Finally, the molecular mass of the oligomers/polymers which formthe nanoparticles according to the invention can be perfectly masteredby adjusting the following preparative conditions:

[0023] the monomer concentration in the organic phase,

[0024] the pH and the molarity of the polymerisation medium,

[0025] the nature and the concentration of the dispersing agent,

[0026] the volume ratio of the aqueous phase (polymerisationmedium)/organic phase,

[0027] the mode of introduction of the organic mixture in the aqueousphase.

[0028] In a 1^(st) aspect therefore, the invention relates to a methodfor the preparation of nanoparticles formed from a random polymer of atleast one compound of formula (I)

[0029] in which

[0030] A represents a

[0031] group;

[0032] R₁ and R₂, identical or different, represent a linear or branchedC₁-C₆ alkyl group;

[0033] n=1, 2, 3, 4 or 5;

[0034] characterised in that the monomer(s) is (are), before thepolymerisation, dissolved in a water-miscible aprotic organic solventforming, with the polymerisation medium, a non-solvent mixture of thepolymer formed.

[0035] In an advantageous aspect, the invention relates to a method forthe preparation of nanoparticles formed from a polymer of a compound offormula (I)

[0036] group;

[0037] R₁ and R₂, identical or different, represent a linear or branchedC₁-C₆ alkyl group;

[0038] n=1, 2, 3, 4 or 5;

[0039] characterised in that before the polymerisation, the monomer isdissolved in a water-miscible aprotic organic solvent forming, with thepolymerisation medium, a non-solvent mixture of the polymer formed.

[0040] According to a particular aspect, the method according to theinvention enables the preparation of nanoparticles having a diameter ofless than 500 nm, preferably between 100 and 500 nm. and an averagemolecular mass (Mw) between about 1000 and 100000, notably between about1000 and 80000. in particular between about 2000 and 80000, preferablybetween about 8000 and 80000.

[0041] In particular, the method according to the invention comprisesthe steps consisting in:

[0042] preparing a solution of at least one compound of formula (I) in awater-miscible aprotic organic solvent.

[0043] adding, with stirring, this organic phase to an aqueouspolymerisation medium at a pH between 4.5 and 10,

[0044] recovering the nanoparticles thus obtained after homogenisationof the mixture and evaporating the organic solvent in vacuo.

[0045] The aqueous polymerisation medium can also be added to theorganic phase which contains the monomer dissolved beforehand, andaccording to another aspect, the method according to the inventioncomprises the steps consisting in

[0046] preparing a solution of at least one compound of formula (I) in awater-miscible aprotic organic solvent,

[0047] adding, with stirring, to this organic phase, an aqueouspolymerisation medium at a pH between 4.5 and 10,

[0048] recovering the nanoparticles thus obtained after homogenisationof the mixture and evaporating the organic solvent in vacuo.

[0049] As illustrated later on in the Examples, the pH of thepolymerisation medium is selected as a function of the molecular mass ofthe polymer that is desired to prepare.

[0050] Advantageously, the mixture of the organic phase and the aqueousmedium is homogenised by continuous stirring for about 30 minutes andthen, optionally, the preparation is completed by distilled water.

[0051] The polymer formed precipitates in the polymerisation medium andcan be recovered by filtration for example. The nanoparticle suspensionthus obtained can then be conditioned and lyophilised.

[0052] The aprotic organic solvent used for dispersing the monomer(s)must be a solvent of said monomer(s) which should also be miscible withwater. This solvent is preferably selected from acetone, acetonitrile,dioxane and tetrahydrofuran. acetone being particularly preferred.

[0053] Preferred aspects of the method are the following:

[0054] the concentration of monomer(s) of formula (I) in the organicsolvent is of the order 30 mg/ml to 150 mg/ml;

[0055] the molarity of the polymerisation medium is of the order of{fraction (1/30)}M to ⅓M;

[0056] volume ratio of the aqueous phase to the organic phase is between3/1 and 20/1, preferably between 3/1 and 15/1.

[0057] Advantageously, the polymerisation medium contains one or moresurfactants or colloid protectors.

[0058] The surfactants can be ionic or non-ionic surfactants forexample. Non-ionic surfactants will preferably be used which areselected from copolymers of polyoxyethylene and polyoxypropylene,poloxamers and polysorbates. As colloid protector agents, polysaccharidederivatives will preferably be used, such as dextrans, hydrosolublecellulose derivatives: polyethylene glycols poly(vinyl alcohol).

[0059] Preferably, the compound polymerised to form the nanoparticlesaccording to the method of the invention is a compound of formula (I) inwhich : A represents a

[0060] group, n=1 and R₁=R₂=ethyl.

[0061] In another preferred aspect, the compound polymerised to form thenanoparticles according to the method of the invention is a compound offormula (I) in which: A represents a

[0062] group, and R₁=R₂=propyl.

[0063] Advantageously, a mixture of compounds of formula (I) in which Ais a

[0064] group as defined above, can also be random polymerised.

[0065] In a 2_(nd) aspect, the invention relates to the nanoparticlesformed from a random polymer of at least one methylidene malonatecompound of formula (I), having a diameter of less than 500 nm,preferably between 100 and 500 nm and an average molecular mass (Mw)between about 1000 and 100000, notably between 1000 and 80000, inparticular between about 2000 and 80000, preferably between about 8000and 80000, obtainable by this method.

[0066] In particular, said nanoparticles, obtainable by this method, areformed from a polymer of a compound of formula (I), have a diameter ofless than 500 nm, preferably between 100 and 500 nm and an Mw betweenabout 1000 and 80000, in particular between about 2000 and 80000,preferably between about 8000 and 80000.

[0067] In a preferred aspect, the invention relates to nanoparticlesformed from a random polymer of at least one compound of formula (I),having a diameter of less than 500 nm. preferably between 100 and 500 nmand an average molecular mass (Mw) between about 8000 and 100000,preferably between about 8000 and 80000.

[0068] In particular, the invention relates to nanoparticles formed froma polymer of a compound of formula (I), having a diameter of less than500 nm, preferably between 100 and 500 nm and an average molecular mass(Mw) between about 8000 and 80000.

[0069] Advantageously, said nanoparticles are formed from a compound offormula (I) in which A represents a

[0070] group, n=1 and R₁=R₂=ethyl.

[0071] In another preferred aspect, said nanoparticles are formed from acompound of formula (I) in which A represents a

[0072] group and R₁=R₂=propyl.

[0073] Advantageously, said nanoparticles can be constituted of a randompolymer of a mixture of compounds of formula (I) in which A is a

[0074] group as defined above.

[0075] According to a further aspect of the invention, saidnanoparticles comprise, in their polymeric network, one or morebiologically active molecules such as mentioned above.

[0076] In fact, in an advantageous aspect of the method according to theinvention, the organic phase (when it is a biologically active moleculewhich is insoluble in water) or the polymerisation medium can containone or more biologically active molecules.

[0077] “Biologically active molecule” is understood as meaning, in anon-limiting way, any molecule or macromolecule which has a prophylacticor curative biological activity, in vitro or in vivo, notably ananti-infectious agent, in particular an antiseptic agent, an antibiotic,an antiviral, an antiparasitic or antimitotic agent, notably ananticancer agent.

[0078] Antibiotic or antiseptic agents which can be used can be, forexample, rifampicin and colistin.

[0079] As antiviral agents, didanosin, ribavirin, zidovudin, acyclovir,ganciclovir, foscarnet, vidarabin and zalcitabin can be cited in anon-limiting way.

[0080] Cis-plastin, 5-fluorouracil or taxol can, for example, be used asanti-cancer agents. Another advantageous antitumor agent is creatinephosphate whose activity is described in the application EP 0 614 366.

[0081] The invention also relates to pharmaceutical compositionscontaining said nanoparticles which comprise one or more biologicallyactive molecules in association with a pharmaceutically acceptablevehicle.

[0082] The compositions according to the invention can be compositionswhich can be administered for example orally, sublingually,subcutaneously, intramuscularly, intravenously, transdermally, locally,rectally, via the pulmonary route, or nasally.

[0083] The suitable forms of administration notably comprise oral forms,such as tablets, gelatine capsules, powders, granules and oral solutionsor suspensions, sublingual and buccal administration forms, as well assubcutaneous, intramuscular, intravenous, intranasal or intraocular andrectal administration forms.

[0084] The invention is illustrated by the Examples below, in which thepreparation of the particles is carried out at ambient temperature(about 21° C.). The size, or diameter, of the nanoparticles was measuredwith a laser diffusion counter (Coulter Electronic Inc., USA). Themolecular mass of the polymers was determined by gel permeationchromatography.

EXAMPLE 1

[0085] 500 mg of1-ethoxycarbonyl-1-ethoxycarbonylmethyleneoxycarbonylethene(Laboratoires UPSA/CARPIBEM, France), already desorbed of SO₂ for 3hours under 25 mbars. are dissolved in 5.55 ml acetone. This solution isthen mixed gradually and under magnetic stirring with 50 ml of anaqueous medium buffered at pH 8 (Na₂HPO₄/KH₂PO_(4.)1/15 M) andcontaining 500 mg of dextran 70 (FLUKA CHEMIE, Switzerland). The almostinstant polymerisation produces a cloudiness of the mixture whichpossesses a Tyndall effect characteristic of colloidal solutions.Stirring is maintained for 30 minutes after the complete introduction ofthe organic phase. Next. 50 ml of distilled water containing 2.5 g ofglucose or trehalose (colloid protectors and cryoprotectors) are addedto the nanoparticle suspension and the mixture is submitted to anevaporation in vacuo so as to remove the acetone and to reduce thevolume of the aqueous suspension to 50 ml. After filtration on filterpaper (pore diameter 5 to 15 μm), the preparation is lyophilised. Asmeasured by laser diffusion, the particles contained in the filtratehave a diameter of 288 nm. The average molecular mass (Mw) of themethylidene malonate constituting the polymer matrix of the particles isevaluated to be 67000 by gel permeation chromatography.

EXAMPLE 2 pH Variation Study

[0086] The experiment is carried out following the technique describedin Example 1, but only varying the pH only of the phosphate buffer. Theresults are given in Table 1 below, in which Mp is the molecular mass ofthe principal species and Mw is the average molecular mass of thepolymer. TABLE 1 pH of the polymerisation medium 4.5 5.0 5.5 6.0 6.5 7.07.5 8.0 size (nm) 280 344 424 423 361 382 313 288 standard 9 9 7 6 9 7 23 deviation +/− nm characteristics of the polymer (Ep) Mp 662 655 65519700 31500 36900 40300 59300 Mw 2080 4740 11140 17600 28900 39000 5320067200

[0087] The results show that the average molecular mass of the polymerswhich constitute the nanoparticles increase regularly with the pH of thepolymerisation medium.

[0088] The gel permeation chromatographic profile of FIG. 1 representsthe distribution of the molecular mass of the polymer prepared at pH 5.5(concentration: 90 mg/ml). A broad peak is observed in 1 whichcorresponds to the species of high average molecular mass (Mw) and anarrow peak is observed in 2 which corresponds to the minor oligomers(major trimers and tetramers).

[0089] The dotted lines limit the analysable portion of thechromatogram. Peak F is that of toluene used as internal standard andthe negative peak correspond to traces of water.

EXAMPLE 3 Study of the Variation of the Monomer Concentration

[0090] The experiment is carried out following the technique describedin Example 1, but by varying only the monomer concentration in acetone.The results are given in Table 2 below: TABLE 2 monomer concentration inthe organic phase (mg/ml) 30 60 90 size (nm) 213 239 288 standarddeviation 2 4 3 +/− nm characteristics of the polymer (Ep) Mp 3150039600 59300 Mw 44700 63000 67200

[0091] The results show that the molecular mass of the principal species(Mp), as well as the average molecular mass (Mw) of the polymers whichconstitute the nanoparticles, increase regularly with the concentrationof the monomer in the organic phase.

EXAMPLE 4

[0092] The experiment is carried out according to Examples 1 to 3 but inreplacing dextran 70 colloid protector with a non-ionic surfactant,Pluronic F68 (BASF Corporation, USA).

[0093] The results are given in Table 3 below. TABLE 3 pH of thepolymerisation medium containing 0.5% Pluronic F 68 4.5 5.0 5.5 6.0 6.57.0 7.5 8.0 Size (nm) 87 80 95 117 122 121 146 153 standard 1 2 2 5 9 13 1 deviation +/− nm characteristics of the polymer (Ep)* Mp 656 1330014800 25600 38600 43700 45300 77800 Mw 5520 9740 12300 23600 33000 5160070900 88900

[0094] The results show, for the same conditions of pH:

[0095] an increase in the molecular mass of the principal species (Mp)and in the average molecular mass (Mw) of the polymers constituting thenanoparticles in the presence of the surfactant with respect to thecolloid protector,

[0096] a decrease in the size of these same nanoparticles in thepresence of the surfactant with respect to the colloid protector.

EXAMPLE 5 Study of the Molarity of the Polymerisation Medium

[0097] According to the method described in Example 1, 500 mg of monomerare dissolved in 16.6 ml acetone are introduced into a phosphate buffer(Na₂HPO₄/KH₂PO₄) of increasing molarity, and further containing 0.5%Pluronic F68.

[0098] The results are given in Table 4 below: TABLE 4 standard size ofthe deviation Mp Mw molarity nanoparticles (nm) (nm) (Ep) (Ep) 0.033 M127 2 15200 12500 0.066 M 123 1 14600 12400 0.133 M 124 1  653  97900.267 M 179 3  660  8690

[0099] The results show a decrease in the average molecular mass (Mw) ofthe polymers which constitute the nanoparticles in proportion to anincrease in the molarity of the medium.

EXAMPLE 6

[0100] Nanoparticles are prepared according to Examples 1 to 3 and arecompared to the nanoparticles prepared according to the method describedby Lescure et al., Pharm. Res. 1994.11, 1270-1276. For this. 100 mg ofmonomer are introduced with stirring in 10 ml of a phosphate buffermedium (Na₂HPO₄/KH₂PO_(4.)1/15 M) of pH 5 to 8.

[0101] The results are given in Table 5 below in which the oligomers aredefined as any molecular species of molecular mass less than or equal to920. TABLE 5 pH of the polymerisation medium containing 1% dextran 705.5 6.0 6.5 7.0 7.5 8.0 Method according size (nm) 260 296 337 335 271322 to Lescure et standard 5 5 10 4 6 6 al., 1994 deviation +/− nm Mp666 660 675 685 15572 13000 Mw 1719 2421 5335 6041* 6759* 7594* %oligomers 43 53 27 38 18 13 yield of 87 79.5 71.5 59.5 21 23nanoparticles obtained % ±5 Method according size (nm) 424 423 361 382313 288 to the invention** standard 7 6 9 7 2 3 deviation +/− nm Mp 65519695 31508 36290 40278 59300 Mw 11138 17569 28918 38997 53181 67201 %oligomers 19 14 8 4 3 2 yield of 82.5 73 84.5 91 85 86.5 nanoparticlesobtained % ±5

[0102] The results show that, for any experimental condition ofidentical pH

[0103] the average molecular mass (Mw) of the polymers constituting thenanoparticles prepared according to Lescure et al. is less than that ofthe polymer obtained according to the method of the invention;

[0104] the contents of the oligomers (trimers-tetramers) constitutingthe polymers are significantly less for the nanoparticles preparedaccording to the method of the invention;

[0105] the yields of polymerisation in the form of nanoparticles arehigher for the method of the invention compared to the method accordingto Lescure et al (the formation of aggregates results in low yields atbasic pH for the method according to Lescure et al.).

[0106] The gel permeation chromatography profile of FIG. 2 representsthe distributions of molecular mass of the polymers prepared at pH 7.5according to the method of the invention on the one hand (trace A), andaccording to the method of Lescure et al on the other (trace B). Apartfrom peak 3 corresponding to toluene, for peak A, a single peak 1 isobserved which corresponds to the principal species (Mp=40278) while fortrace B, the presence of a significant peak 2 is observed also whichcorresponds to the oligomers (trimers and tetramers).

EXAMPLE 7

[0107] 50 ml of an aqueous medium buffered at pH 5; 6.5 or 8(Na₂HPO₄/KH₂PO₄ 1/15 M) and containing 0.5% of Pluronic F68 (BASFCorporation, USA) are added gradually and with magnetic stirring to 5.55ml of a solution of 500 mg of1-ethoxycarbonyl-1-ethoxycarbonylmethyleneoxy-carbonylethene monomer(LABORATOIRES UPSA/CARPIBEM, France), already desorbed of SO₂ for 3hours under 25 mbars in 5.55 ml of acetone. The stirring is maintainedfor 16 hours for the tests at pH 5 and 6.5 or for 30 minutes for thetest at pH 8 after the complete introduction of the organic phase. Next,50 ml of distilled water containing 2.5 g of glucose or trehalose(colloid protectors and cryoprotectors) are added to the nanoparticlesuspension and the mixture is submitted to evaporation in vacuo so as toremove the acetone and to reduce the volume of the aqueous suspension to50 ml. After filtration on filter paper (pore diameter 5 to 15 μm), thepreparation is lyophilised. The diameter of the particles contained inthe filtrate is measured by laser diffusion. The average molecular mass(Mw) of the methylidene malonate constituting the polymer matrix of theparticles is evaluated by gel permeation chromatography.

[0108] The results are given in Table 6 below, in which Mp is themolecular mass of the principal species and Mw is the average molecularmass of the polymer.

[0109] The yield is determined by the ratio of the amount of monomerintroduced into the reaction medium and the amount of polymerconstituting the nanoparticles. TABLE 6 pH of the polymerisation medium5.0 6.5 8.0 size (nm) 848 394 754 standard deviation 36 32 34 +/−nmcharacteristics of the polymer (Ep) Mp 312 24300 26500 Mw 6450 2010020100 yield % 59 57 30 standard deviation 5.1 4.6 4.2

EXAMPLE 8 Use of Different Solvents

[0110] The experiment is carried out following the method of Example 1,but using acetone, acetonitrile or tetrahydrofuran (THF) as solvent ofthe monomer.

[0111] The results are given in Table 7 below. TABLE 7 Average particleSolvent size (nm) yield (%) Mw Acetone 253 74 54 100 Acetonitrile 197 6931 700 THF 191 70 30 300

EXAMPLE 9 Study of the Water/Solvent Volume Ratio

[0112] The experiment is carried out following the method of Example 1,but varying the water/acetone volume ratio.

[0113] The results are given in Table 8 below: TABLE 8 Water/solventvolume ratio 4.5/1 9/1 18/1 size (nm) 241 288 334 yield (%) 74 74 85characteristics of the polymer Mp 62100 59300 33100 Mw 42000 67200 24600

EXAMPLE 10 Implementation of the Method at pH 10.

[0114] The tests were carried out in an aqueous medium at pH=10 in thepresence either of a surfactant or a colloid protector and this, eitherfollowing the method of Example 1 or following the method of Example 7.

[0115] 1) test 1

[0116] 100 mg of1-ethoxycarbonyl-1-ethoxycarbonylmethyleneoxycarbonyl-ethene monomer aredissolved in 1 ml of acetone.

[0117] This solution is then added gradually and with magnetic stirringinto 10 ml of an aqueous medium at pH=10 and containing 100 mg ofDextran 70.

[0118] The polymerisation is instantaneous. The stirring is maintainedfor 30 minutes after the introduction of the whole of the organic phase.Next, 10 ml of distilled water are added to the nanoparticle suspension,and the mixture is submitted to an evaporation in vacuo so as to removethe acetone. The medium is then centrifuged (v=10 000 rpm. 10 min at 4°C.).

[0119] 2) test 2

[0120] The experimental protocol is identical to that of test 1 but byreplacing Dextran 70 with Pluronic F68.

[0121] 3) test 3

[0122] 10 ml of an aqueous medium at pH=10 containing 100 mg of Dextran70 are added gradually with magnetic stirring into an organic phaseconstituted of 100 mg of monomer and 1 ml of acetone. The polymerisationis instantaneous. The stirring is maintained for 30 minutes after theintroduction of the whole of the aqueous phase. Next, 10 ml of distilledwater are added to the nanoparticle suspension and the mixture issubmitted to an evaporation in vacuo so as to remove the acetone. Themedium is then centrifuged (v=10 000 rpm. 10 min at 4° C.).

[0123] 4) test 4

[0124] The experimental protocol is identical to that of test 3 but theDextran 70 is replaced with Pluronic F68. After centrifugation, thenanoparticles contained in the plug are analysed by steric exclusionchromatography to determine their weight average molecular mass (Mw).

[0125] The results are given in the Table 9 below. TABLE 9 Mw Particlesize (nm) Test 1 8 800 240 Test 2 6 900 245 Test 3 1 400 316 Test 4 1850 333

EXAMPLE 11

[0126] The experiment is carried out following the polymerisationtechnique described in Example 1, but using 1,1-propoxycarbonylethene(Laboratoires UPSA/CARPIBEM, France) hereinafter referred to as MM 3.3alone or in a mixture with the1-ethoxycarbonyl-1-ethoxycarbonylmethyleneoxy-carbonylethene monomer(Laboratoires UPSA/CARPIBEM. France), hereinafter referred to as MM2.1.2. The results are given in Table 10 below, in which Mp is themolecular mass of the principal species and Mw is the average molecularmass of the polymer. TABLE 10 Ratio MM 3.3/MM 2.1.2 100/0 75/25 50/5025/75 Size 123 223 298 155 Yield (%) 77 73 80 78 Characteristics of thepolymer Mp 44764 92090 37467 21727 Mw 44122 89793 37467 21727

EXAMPLE 12 Preparation of Nanoparticles Containing Rifampicin

[0127] 5 mg of rifampicin base (Sigma) are dissolved in 1 ml of acetoneto which 90 mg of 1-ethoxycarbonyl-1-ethoxycarbonylmethyleneoxy-carbonylethene monomer (LABORATOIRES UPSA/CARPIBEM. France)are added, beforehand desorbed of SO₂ for 3 hours under 25 mbars. Withthe aid of a glass pipette, this solution is then added gradually andwith constant stirring (750 rpm) to 9 ml of aqueous medium buffered atpH 6.0 with the aid of a phosphate buffer (Na₂HPO₄/KH₂PO₄ 0.066 M) andcontaining 90 mg of dextran 70 (1% w/v). After 18 hours ofpolymerisation at 20° C., 9 ml of distilled water containing 5% ofD-glucose are added with stirring to the nanoparticle suspension, themixture is then submitted to an evaporation in vacuo with the aid of aRotavapor (20° C., 25 mbars) so as to remove the acetone and to reducethe volume of the aqueous suspension to 9 ml. The preparation is thenlyophilised: freezing takes place at −30° C. and sublimation at +20° C.for 36 hours at a pressure of 0.05 mbar.

[0128] The size of the nanoparticles and the rifampicin concentrationare measured before and after lyophilisation. The size is measured bylaser diffusion. The determination of the rifampicin is carried out byhigh performance liquid chromatography coupled to a spectrophotometer.The mobile phase is composed of a mixture of methanol/0.05 M ammoniumacetate (65:35). the pH is adjusted to 7.3, the flow rate is fixed at 1ml/min and the absorption is read at 254 nm. The content of rifampicinwhich is not bound to the nanoparticles is measured in the supernatantobtained after ultracentrifugation of the nanoparticle suspension (80000g. 1 h at 4° C.). The amount of rifampicin bound to the nanoparticlescorresponds to the fraction present in the plug, which is dissolved inTHF before proceeding with the direct rifampicin determination.

[0129] The following results are obtained:

[0130] size of the nanoparticles containing rifampicin: 266±63 nm beforelyophilisation and 282±54 nm after lyophilisation;

[0131] percentage binding of rifampicin : 8.5±0.5% before and afterlyophilisation.

EXAMPLE 13 Preparation of Nanoparticles Containing Colistin

[0132] The experiment is carried out in the same way as in Example 12,but the active principle being hydrosoluble, it is incorporated in thepolymerisation medium at a concentration of 0.5 mg/ml before addition ofthe organic phase. The size of the nanoparticles containing colistinmeasured by laser diffusion is 282±65 nm after evaporation and 283±26 nmafter conservation at +4° C. for 4 days. Determined according to thegelose diffusion technique (S. P. Gotoff et al., Antimicrob. AgentsChemother, 1962, 107-113), colistin is found at the concentration of 15μg/ml in the supernatant obtained after ultracentrifugation of thenanoparticle suspension (80000 g, 1 hour at 4° C.): the fraction whichis not bound to the nanoparticles is then evaluated at 3% of the totalamount of colistin added.

EXAMPLE 14

[0133] Preparation of nanoparticles containing azidothymidine (AZT)(Sigma Aldrich Chimie, France).

[0134] 240 mg of1-ethoxycarbonyl-1-ethoxycarbonylmethyleneoxycarbonyl-ethene monomer(Laboratoires UPSA/CARPIBEM. France), already desorbed of SO₂ for 3hours under 25 mbars, are dissolved in 2.5 ml acetone. With the aid of apropipette, this solution is then gradually added and with constantstirring to 22.5 ml of aqueous medium buffered at pH 8.0 with the aid ofa phosphate buffer (Na₂HPO₄/KH₂PO₄ 0.066 M) and containing 225 mg ofdextran 70 (1% w/v), as well as the hydrosoluble active principle at aconcentration of 0.53 mg/ml. After 18 hours' polymerisation at 20° C.,22.5 ml of demineralised water containing 5% of D-glucose are added withstirring to the nanoparticle suspension, the mixture is then submittedto an evaporation in vacuo with the aid of a Rotavapor (20° C., 25mbars) so as to remove the acetone and to reduce the volume of theaqueous suspension to 39.0 ml. The preparation is then lyophilised;freezing takes place at −30° C. and sublimation at +20° C. for 36 hoursat a pressure of 0.05 mbar.

[0135] The size of the nanoparticles containing AZT measured by laserdiffusion is 255±63 nm before lyophilisation. The content of AZT in thesupernatant after centrifugation of the nanoparticle suspension (12000rpm, 1 hour at 4° C.) is determined by UV spectrophotometry at 266 nm. Aconcentration of 98 μg/ml is obtained: the fraction which is not boundto the nanoparticles is therefore evaluated to be 31.9% of the totalamount of AZT added. The fraction of AZT bound to the nanoparticles istherefore 68.1%.

EXAMPLE 15 Preparation of Nanoparticles Containing Creatine Phosphate(Boehringer Mannheim).

[0136] The encapsulation of creatine phosphate is carried out accordingto the technique of Example 14. The size of the nanoparticles containingcreatine phosphate measured by laser diffusion is 275±260 nm beforelyophilisation. The determination of the creatine phosphate is carriedout by high performance liquid chromatography coupled to aspectrophotometer. The mobile phase is composed of a phosphate buffer(KH₂PO_(4.) 0.05 M) adjusted to pH 3.3. The flow rate is fixed at 2ml/min and the absorption is read at 200 nm.

[0137] The content of creatine phosphate which is not bound to thenanoparticles is measured in the supernatant obtained aftercentrifugation of the nanoparticle suspension (12000 rpm, 1 hour at 4°C.). The creatine phosphate is found at a concentration of 463 μg/ml inthe supernatant: the fraction which is not bound to the nanoparticles istherefore evaluated at 81% of the total amount of creatine phosphateadded. The fraction of creatine phosphate bound to the nanoparticles istherefore 19%.

EXAMPLE 16 Preparation of Nanoparticles Containing 5-fluorouracile(5-FU)

[0138] The encapsulation of 5-FU (Sigma Aldrich Chimie, France) iscarried out according to the technique of Example 14. The size of thenanoparticles containing the 5-FU measured by laser diffusion is 516±88nm before lyophilisation. Determined by UV spectrophotometry at 266 nm,the 5-EU is found at a concentration of 70 μg/ml in the supernatantobtained after centrifugation of the nanoparticle suspension (12 000rpm, 1 hour at 4° C.): the fraction which is not bound to thenanoparticles is therefore evaluated at 23.3% of the total amount of5-FU added. The fraction of 5-FU bound to the nanoparticles is therefore76.7%.

1. A method for the preparation of nanoparticles formed from a randompolymer of at least one compound of formula (I)

group; R₁ and R₂, identical or different, represent a linear or branchedC₁-C₆ alkyl group; n=1, 2, 3, 4 or 5; characterised in that themonomer(s) is (are), before the polymerisation, dissolved in awater-miscible aprotic organic solvent forming, with the polymerisationmedium, a non-solvent mixture of the polymer formed.
 2. The methodaccording to claim 1 for the preparation of nanoparticles formed from apolymer of a compound of formula (I)

group; R₁ and R₂, identical or different, represents a linear orbranched C₁-C₆ alkyl group; —n=1, 2, 3, 4 or 5, characterised in thatbefore the polymerisation, the monomer is dissolved in a water-miscibleaprotic organic solvent forming, with the polymerisation medium, anon-solvent mixture of the polymer formed.
 3. The method according toone of claims 1 or 2, for the preparation of nanoparticles having adiameter of less than 500 nm. preferably between 100 and 500 nm, and anaverage molecular mass (Mw) between about 1000 and 100000, notablybetween about 1000 and 80000, in particular between about 2000 and80000, preferably between about 8000 and
 80000. 4. The method accordingto any one of claims 1 to 3 , characterised in that it comprises thesteps consisting in: preparing a solution of at least one compound offormula (I) in a water-miscible aprotic organic solvent, adding, withstirring, to this organic phase, an aqueous polymerisation medium at apH between 4.5 and 10, recovering the nanoparticles thus obtained afterhomogenisation of the mixture and evaporating the organic solvent invacuo.
 5. The method according to any one of claims 1 to 3 ,characterised in that it comprises the steps consisting in: preparing asolution of at least one compound of formula (I) in a water-miscibleaprotic organic solvent, adding, with stirring, to this organic phase,an aqueous polymerisation medium at a pH between 4.5 and 10, recoveringthe nanoparticles thus obtained after homogenisation of the mixture andevaporating the organic solvent in vacuo.
 6. The method according to anyone of claims 1 to 5 , characterised in that the aprotic organic solventis selected from acetone, acetonitrile, dioxane and tetrahydrofuran. 7.The method according to any one of claims 1 to 6 , characterised in thatthe concentration of compound(s) of formula (I) in the organic solventis of the order of 30 mg/ml to 150 mg/ml.
 8. The method according to anyone of claims 1 to 7 , characterised in that the molarity of thepolymerisation medium is of the order of {fraction (1/30)}M to ⅓M. 9.The method according to any one of claims 1 to 8 , characterised in thatthe polymerisation medium contains one or more surfactants or colloidprotectors.
 10. The method according to claim 9 , characterised in thatthe surfactants are non-ionic surfactants selected from copolymers ofpolyoxyethylene and polyoxypropylene, poloxamers and polysorbates. 11.The method according to claim 9 or 10 , characterised in that thecolloid protector agents are selected from dextrans, hydrosolublecellulose derivatives, polyethylene glycols and poly(vinyl alcohol). 12.The method according to any one of claims 1 to 11 , characterised inthat the organic phase or the polymerisation medium contains one or morebiologically active molecules.
 13. The method according to any one ofclaims 1 to 12 , characterised in that the polymerised compound is acompound of formula (I) in which A represents a

group, R₁=R₂=ethyl and n=1.
 14. The method according to any one ofclaims 1 to 12 , characterised in that the polymerised compound is acompound of formula (I) in which A represents a

group and R₁=R₂=propyl.
 15. The method according to any one of claims 1to 12 , characterised in that a mixture of compounds of formula (I) inwhich A is a

group as defined in claim 1 , is random polymerised.
 16. Nanoparticlesformed from a random polymer of at least one methylidene malonatecompound of formula (I)

group; R₁ and R₂, identical or different, represents a linear orbranched C₁-C₆ alkyl group; n=1, 2, 3, 4 or 5, having a diameter of lessthan 500 nm, preferably between 100 and 500 nm and an average molecularmass (Mw) between about 1000 and 100000, notably between 1000 and 80000,in particular between about 2 000 and 80 000, preferably between about8000 and 80000, obtainable by the method according to any one of claims1 to 15 .
 17. Nanoparticles formed from a polymer of a methylidenemalonate compound of formula (I)

group; R₁ and R₂, identical or different, represents a linear orbranched C₁-C₆ alkyl group; n=1, 2, 3, 4 or 5, having a diameter of lessthan 500 nm. preferably between 100 and 500 nm and an average molecularmass (Mw) between about 1000 and 80000, in particular between about 2000 and 80 000, preferably between about 8000 and 80000, obtainable bythe method according to any one of claims 1 to 15 .
 18. Nanoparticlesformed from a random polymer of at least one compound of formula (I)

group; R₁ and R₂, identical or different, represents a linear orbranched C₁-C₆ alkyl group; n=1, 3, 4 or 5, having a diameter of lessthan 500 nm, preferably between 100 and 500 nm and an average molecularmass (Mw) between about 8000 and 100000, preferably between about 8000and
 80000. 19. Nanoparticles formed from a polymer of a compound offormula (I)

group; R₁ and R₂, identical or different, represents a linear orbranched C₁-C₆ alkyl group; n=1, 2, 3, 4 or 5, having a diameter of lessthan 500 nm, preferably between 100 and 500 nm and an average molecularmass (Mw) between about 8000 and
 80000. 20. The nanoparticles accordingto one of claims 18 or 19, formed from a polymer of a compound offormula (I) in which A is a

group, n−1, R₁=R₂=ethyl.
 21. The nanoparticles according to one ofclaims 18 or 19, formed from a polymer of a compound of formula (I) inwhich A is a

group and R₁=R₂=propyl.
 22. The nanoparticles according to any one ofclaims 16 to 21 , characterised in that they comprise one or morebiologically active molecules.
 23. Pharmaceutical composition containingnanoparticles according to claim 22 as active principle in associationwith a pharmaceutically acceptable vehicle.